The muscular dystrophies, many caused by mutations in genes encoding proteins of the dystrophin complex, are among the most prevalent and devastating human diseases. No cures exist and current treatments that slow muscle degeneration are largely ineffective. The goal of this application is to apply basic knowledge of several muscular dystrophies to developing therapeutic approaches. In project 1, Jeffrey Chamberlain will isolate alternative types of myogenic stem cells, correct the primary genetic lesion in these cells by gene transfer, and explore the use of such cells for transplantation into syngeneic, dystrophic mice. He will generate myogenic stem cells from dystrophic muscle fibroblasts and explore their ability to generate new muscle tissue in vitro and in vivo and explore the therapeutic use of pericytes isolated from dystrophic muscle. In project 2, Stephen Tapscott will expand the cell therapy approach by examining muscle cell transplantation in the canine model of muscular dystrophy. Enhancement of migration and engraftment of transplanted donor cells will be explored by modulating signaling pathways and extracellular matrix components and genetic manipulations. Finally, specific muscle derived cell populations will be compared for their ability to reconstitute canine skeletal muscle in vivo. In project 3, Stephen Hauschka will modify muscle-specific regulatory cassettes to provide high expression in human muscle cultures. Modified cassettes will then be tested in vivo for expression of therapeutic proteins after AAV and Lentiviral delivery to human muscle xenografts in immunodeficient mice. Clonal satellite cell assays and analysis of human muscle fiber regeneration following xenograft injury will determine whether the satellite cell pool has been stably transduced. In project 4, Stanley Froehner will study a new compensatory gene, NPC1, which markedly reduces the severity of the dystrophic phenotype in mdx mouse muscle. The mechanism of NPC1 phenotype amelioration and its applicability to LGMDs will be studied. Two core facilities will serve the participating laboratories. PUBLIC HEALTH RELEVANCE: Results from this integrated project will lead to new therapeutic approaches for DMD and other muscular dystrophies. PROJECT 1 Principal Investigator: Jeffrey S. Chamberlain Title: Dystrophin Delivery to Muscle via Myogenic Precursors Description (provided by applicant): Dystrophin delivery to muscle via myogenic progenitors (Chamberlain, JS, P.I.)- Skeletal muscles in Duchenne muscular dystrophy (DMD) patients undergo cycles of necrosis and regeneration leading to loss of muscle fibers and replacement with adipose and connective tissue. Regeneration is supported by activation and recruitment of satellite cells, but the regenerative capacity of dystrophic muscles decreases over time due to poorly understood changes in the muscle microenvironment perhaps coupled with the onset of proliferative senescence. Loss of regenerative capacity is a major contributing factor to decreasing muscle strength, and leads to profound muscle wasting. One approach to treating DMD and other muscle wasting disorders is to identify and/or generate myogenic progenitor/stem cells either from donors or patients, expand them in vitro or in vivo and use these cells to at least partially restore muscle mass and regenerative capacity. However, satellite cell based therapies have so far failed and there is a great need for alternative stem cells with better potential to support muscle regeneration following transplantation. The goals of this project are to isolate and generate alternative stem cells from dystrophic muscles, to correct the genetic lesion in these cells by gene transfer, and to explore autologous transplantation into dystrophic mice. Towards these goals we have developed a robust system for generating lentiviral (LV) vectors that are able to permanently transduce a variety of dividing and non-dividing cells, including myoblasts, satellite cells, fibroblasts, pericytes and mesoangioblasts. LV (and AAV) vectors can also be directly injected into muscles (or blood vessels in the case of AAV) of mice, leading to gene expression in satellite cells and myofibers for the lifespan of a mouse. We have shown that fibroblasts from dystrophic mice can be genetically corrected and modified to form myogenic progenitors after transplantation into mdx hosts. We have also been exploring the isolation and genetic modification of pericytes for autologous cell therapy of muscular dystrophy. We propose to use fibroblasts directly and indirectly via induction of pluripotent stem (iPS) cells, and to compare their efficacy with that of pericytes upon transplantation into mouse models of DMD. Public Health Relevance: Our goal is to develop methods that could be used to treat the muscular dystrophies (MDs). Stem cell therapy is a promising approach, but many limitations prevent its implementation at present. Our studies are designed to identify an accessible source of muscle stem cells that could be used for autologous stem cell therapies, and to genetically manipulate those cells to produce the protein missing in common MDs.